The present invention relates generally to optical detection systems, and in particular to a multi-channel detection system for the real-time detection of a plurality of different analytes in a fluid sample.
There are many applications in the field of chemical processing in which it is desirable to precisely control the temperature of reaction mixtures (e.g., biological samples mixed with chemicals or reagents), to induce rapid temperature transitions in the mixtures, and to detect target analytes in the mixtures. Applications for such heat-exchanging chemical reactions may encompass organic, inorganic, biochemical and molecular reactions, and the like. Examples of thermal chemical reactions include nucleic acid amplification, thermal cycling amplification, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication, enzyme kinetic studies, homogeneous ligand binding assays, and more complex biochemical mechanistic studies that require complex temperature changes.
A preferred detection technique for chemical or biochemical analysis is optical interrogation, typically using fluorescence or chemiluminescence measurements. For ligand-binding assays, time-resolved fluorescence, fluorescence polarization, or optical absorption are often used. For PCR assays, fluorescence chemistries are often employed.
Conventional instruments for conducting thermal reactions and for optically detecting the reaction products typically incorporate a block of metal having as many as ninety-six conical reaction tubes. The metal block is heated and cooled either by a Peltier heating/cooling apparatus or by a closed-loop liquid heating/cooling system in which liquid flows through channels machined into the block. Such instruments incorporating a metal block are described in U.S. Pat. No. 5,038,852 to Johnson, U.S. Pat. No. 5,333,675 to Mullis, and U.S. Pat. No. 5,475,610 to Atwood.
These conventional instruments have several disadvantages. First, due to the large thermal mass of a metal block, the heating and cooling rates in these instruments are limited to about 1xc2x0 C./sec resulting in longer processing times. For example, in a typical PCR application, fifty cycles may require two or more hours to complete. With these relatively slow heating and cooling rates, it has been observed that some processes requiring precise temperature control are inefficient. For example, reactions may occur at the intermediate temperatures, creating unwanted and interfering side products, such as PCR xe2x80x9cprimer-dimersxe2x80x9d or anomalous amplicons, which are detrimental to the analytical process. Poor control of temperature also results in over-consumption of reagents necessary for the intended reaction.
Another disadvantage of these conventional instruments is that they typically do not permit real-time optical detection or continuous optical monitoring of the chemical reaction. For example, in the Perkin Elmer 7700 (ATC) instrument, optical fluorescence detection is accomplished by guiding an optical fiber to each of ninety-six reaction sites in a metal block. A central high power laser sequentially excites each reaction site and captures the fluorescence signal through the optical fiber. Since all of the reaction sites are sequentially excited by a single laser and since the fluorescence is detected by a single spectrometer and photomultiplier tube, simultaneous monitoring of each reaction site is not possible.
Some of the instrumentation for newer processes requiring real-time optical monitoring of a chemical reaction has only recently become available. One such instrument is the MATCI device disclosed by Northrup et al in U.S. Pat. No. 5,589,136. This device uses a modular approach to PCR thermal cycling and optical analysis. Each chemical reaction is performed in its own silicon sleeve and each sleeve has its own associated optical excitation source and fluorescence detector. Using a light-emitting diode (LED) and a solid-state detector, real-time optical data is obtained from a compact, low-power module. The device includes only one light source and one detector for each module, however, so that the simultaneous detection of multiple analytes is not possible.
Another analysis instrument is available from Idaho Technologies and described by Wittwer et al. in xe2x80x9cThe LightCycler(trademark): A Microvolume Multisample Fluorimeter with Rapid Temperature Controlxe2x80x9d, BioTechniques, Vol. 22, pgs. 176-181, January 1997. The instrument includes a circular carousel with a stepper motor for holding up to twenty-four samples and for sequentially positioning each of the samples over an optics assembly. The temperature of the samples is controlled by a central heating cartridge and a fan positioned in a central chamber of the carousel.
In operation, the samples are placed in capillaries which are held by the carousel, and each sample is interrogated through a capillary tip by epi-illumination. The light source is a blue LED that is reflected off a first dichroic filter towards the sample. Light is focused to and collected from the capillary tip by an epi-illumination lens. Light emitted from the capillary tip passes through the first dichroic filter, is filtered by one or more additional dichroic filters, and is focused to photodiodes for detection.
Although this instrument permits detection of multiple analytes in a sample undergoing chemical reaction, it has several disadvantages. First, the illumination beams and the emitted light beams have relatively short optical path lengths through the sample volume and share the same path below the capillary tip. This may cause fluorescent emissions from the sample to be weak, leading to poor optical detection sensitivity. Second, the instrument only provides illumination light in one excitation wavelength range. Different fluorescent dyes have different optimal excitation wavelength ranges, however, so that the instrument cannot provide excitation beams in the optimal excitation wavelength range for each of multiple fluorescent dyes in the reaction fluid. Third, the use of dichroic filters may significantly decrease the optical sensitivity of the instrument. Each dichroic filter decreases the intensity of the emitted light by about half, so that the emitted light beams may be weak by the time they reach the detectors. For these reasons, the instrument may exhibit poor sensitivity in detecting fluorescently labeled analytes in the samples.
U.S. Pat. No. 5,675,155 issued to Pentoney et al. discloses another detection system for sequentially and repetitively scanning a plurality of sample volumes and for detecting radiation emitting from each of the samples. The system includes a plurality of coplanar side-by-side capillaries each containing a sample volume. The system also includes an electromagnetic radiation source, a mirror aligned to receive and reflect electromagnetic radiation, a scanner for moving the mirror, a filter wheel for filtering electromagnetic radiation collected from the samples, and a detector aligned to receive the filtered radiation. The sample volume in each capillary column contains fluorescently-labeled samples separated on an electrophoretic medium.
In operation, the radiation source, preferably a laser, directs an excitation beam onto the mirror. The reflected excitation beam passes through a focusing lens and onto a sample volume of a first capillary within the capillary array. Fluorescence emission radiation from the sample is collected and passed through a first filter of the filter wheel which is selected to block light at the wavelength of the laser source and to transmit fluorescence emitted by a first fluorescent dye in the sample volume. Fluorescence transmitted through the first filter is then detected by the detector. A motor then rotates the filter wheel to bring a second filter into the fluorescence emission beam. The second filter transmits fluorescence emitted by a second fluorescence dye, and the fluorescence is measured by the detector. The same process is repeated with third and fourth filters of the filter wheel to measure the fluorescent emission of third and fourth dyes in the sample volume. The entire four-step operation is then performed sequentially and repeatedly with each capillary column in the array.
Although this system permits the detection of multiple fluorescent dyes in a sample volume, it has several disadvantages in its use of a moving mirror and a rotating filter wheel. These moving parts typically result in a high cost of the optical system, high maintenance requirements, low reliability, high power consumption, and potential vibratory interference with the optical measurements.
The present invention overcomes the disadvantages of the prior art by providing an improved system for thermally controlling and optically interrogating reaction mixtures (e.g., biological samples mixed with chemicals or reagents). In contrast to the prior art devices described above, the system of the present invention provides excitation light to each mixture in multiple, distinct excitation wavelength ranges. This ensures that the optimal excitation wavelength range is provided for each of a plurality of analytes in the mixture having different fluorescent, phosphorescent, chemiluminescent, or electrochemiluminescent labels. In addition, the system permits the simultaneous, real-time detection of multiple analytes in the mixture without requiring any moving parts, e.g., carousels or optical filter wheels. Because it has no moving parts, the system of the present invention typically has a lower cost, lower maintenance requirements, higher reliability, and lower power consumption than the prior art devices described above.
The system of the present invention also overcomes the disadvantages of the prior art by providing for extremely rapid and accurate temperature changes of the reaction mixtures. Such tight control of temperature inhibits side reactions, such as the formation of unwanted bubbles or the degradation of components at certain temperatures, that would otherwise interfere with optical detection and analysis. The system is therefore useful in thermally sensitive chemical processes, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication, enzyme kinetic studies, homogeneous ligand binding assays, and more complex biochemical mechanistic studies that require complex temperature changes.
In a preferred embodiment, the invention provides a system for independently thermally controlling and optically interrogating a plurality of reaction mixtures. The system includes a plurality of reaction vessels, each of the vessels having a reaction chamber for holding one of the mixtures. Each of the vessels also includes first and second optically transmissive walls defining a portion of its chamber. The optically transmissive walls are angularly offset from each other to allow optical excitation of the mixture through the first wall and optical detection of labeled analytes through the second wall.
The system also includes a corresponding plurality of heat-exchanging modules for receiving the vessels. Each module includes a pair of opposing thermal plates positioned to receive one of the vessels between them. At least one of the plates, and preferably both of the plates, has a heating element coupled thereto for heating the reaction mixture contained in the vessel. Each module also includes first and second optics assemblies positioned such that when the vessel is placed between the plates, the first and second optics assemblies are in optical communication with the first and second optically transmissive walls of the vessel, respectively.
The first optics assembly includes a first housing having a first optical window, and at least two light sources for transmitting excitation beams to the reaction mixture through the first window. The first optics assembly also includes a first set of filters for filtering the excitation beams such that each of the beams transmitted to the reaction mixture has a substantially distinct excitation wavelength range. In operation, the light sources are sequentially activated to excite different fluorescent, phosphorescent, chemiluminescent, or electrochemiluminescent labels in the reaction mixture. The light sources and the first set of filters are rigidly fixed in the first housing.
The second optics assembly includes a second housing having a second optical window for receiving light emitted from the vessel. The second optics assembly also includes at least two detectors, preferably photodiodes, for detecting the emitted light and a second set of filters for separating the emitted light into at least two emission wavelength ranges and for directing the emitted light in each of the emission wavelength ranges to a respective one of the detectors. The detectors and the second set of filters are rigidly fixed in the second housing.
In the preferred embodiment, the first optics assembly of each module includes at least four light sources arranged with the first set of filters for transmitting the excitation beams in at least four excitation wavelength ranges, and the second optics assembly of each module includes at least four detectors arranged with the second set of filters for detecting emitted light in at least four emission wavelength ranges. The system thus includes at least four separate optical channels for detecting up to four different analytes in each reaction mixture. Also in the preferred embodiment, the system includes a base instrument for receiving the heat-exchanging modules. The base instrument includes processing electronics for independently controlling the operation of each module. The system also preferably includes a computer programmed to control the processing electronics in the base instrument.
Although it is presently preferred to position all of the light sources. in the first optics assembly and all of the detectors in the second optics assembly, it is also possible to include both one or more light sources and one or more detectors in each of the optics assemblies. According to a second embodiment of the invention, the first optics assembly comprises a first housing having a first optical window. The first optics assembly also includes a first light source for transmitting a first excitation beam to the reaction mixture through the first window and a first detector for receiving light emitted from the chamber through the first window. The first optics assembly further includes a first set of filters arranged in the first housing for filtering portions of the first excitation beam outside of a first excitation wavelength range, for filtering portions of the emitted light outside of a first emission wavelength range, and for directing the emitted light in the first emission wavelength range to the first detector. The first light source, the first set of filters, and the first detector are rigidly fixed in the first housing.
Also according to the second embodiment, the second optics assembly comprises a second housing having a second optical window. The second optics assembly also includes a second light source for transmitting a second excitation beam to the reaction mixture through the second window and a second detector for receiving light emitted from the chamber through the second window. The second optics assembly further includes a second set of filters arranged in the second housing for filtering portions of the second excitation beam outside of a second excitation wavelength range different than the first excitation wavelength range, for filtering portions of the emitted light outside of a second emission wavelength range different than the first emission wavelength range, and for directing the emitted light in the second emission wavelength range to the second detector. The second light source, the second set of filters, and the second detector are rigidly fixed in the second housing, so that the optical system has no moving parts. In the second embodiment, each optics assembly may optionally include an additional detector and filter to provide four optical detection channels for detecting up to four different analytes in each reaction mixture.
A more complete understanding of the system of the present invention may be gained upon consideration of the following description and accompanying drawings.